【作者】 沈燕；

【导师】 熊思东；

【作者基本信息】 复旦大学 ， 免疫学， 2003， 博士

【摘要】 病毒性心肌炎（viral myocarditis，VMC）是一临床常见心血管系统疾病。近年来在我国VMC的发病呈上升趋势，并且已成为青少年不明原因猝死的主要原因之一。不仅如此，慢性VMC还可导致扩张性心脏病的发生，严重危害生命健康，加强研究力度很有必要。迄今，VMC的治疗尚无有效的办法，其根本原因在于VMC的发病机制还不完全清楚。因此，阐明VMC的发病机制具有重要的理论和实践意义。病毒感染是VMC重要病因之一，其中由B3型柯萨奇病毒（coxsackievirus group B type 3,CVB3）感染引起的VMC占发病人数的一半以上。研究表明CVB3不仅可通过其编码的Pro2A改变心肌细胞骨架发挥直接损伤心肌细胞的作用，更为重要的是，CVB3还可能通过诱生非特异性和特异性免疫应答反应引起心肌细胞的损害，发挥间接致病作用。现已知，CVB3感染导致心肌细胞免疫损伤的机制与大量炎症细胞浸润有关，炎症细胞在心肌组织中的浸润是VMC特征性的病理学改变之一。但迄今为止，国内外均未见有关CVB3感染是如何启动炎症细胞浸润以及浸润的炎症细胞如何滞留在组织局部发挥免疫损伤作用的报道，这些问题的解决对阐明VMC发病机制很有价值。通常，炎症细胞的浸润是细胞与细胞、细胞与分子之间相互作用的结果，是一多因素、多步骤的复杂过程。炎症细胞在炎症信号的介导和不同的细胞粘附分子的作用下，以钙依赖和非依赖的方式经过滚动、黏附、外渗等步骤，最终聚集在感染局部。研究表明趋化因子（chemokines, ChKs）在提供炎症细胞迁移信号并使之活化、启动炎症细胞定向移动并使之准确定位中扮演着至关重要的角色。我们前期的研究发现，乙肝病毒感染肝细胞后，可导致肝细胞中趋化因子LARC的表达水平改变，这种改变影响杀伤性T细胞（CTL）的浸润。鉴于以上认识，我们推测CVB3感染可能改变心肌组织中趋化因子的表达，改变了表达的趋化因子启动炎症细胞在心肌组织中的浸润和聚集，从而导致VMC的发生。为此，本研究中我们通过小鼠VMC体内外模型，以RT-PCR定性和实时定量分析19种代表性ChKs的表达变化，从中寻找在VMC发病中可能有意义的ChKs。在此基础上，进一步细致研究CVB3感染量、感染时间、活性状态等对在VMC发病中可能有意义的ChKs表达水平的影响。再通过增加心肌组织中ChKs的表达量和干预ChKs的作用，从正反两个方面研究ChKs在VMC发病中的作用及其机制，为VMC发病机制的研究和生物学防治提供新的理论基础和实验依据。更多还原

【Abstract】 ROLE AND MECHANISMS OF MONOCYTE CHEMOATTRACTANT PROTEIN-1(MCP-1) IN PATHOGENESIS OF VIRAL MYOCARDITIS Viral myocarditis (VMC) is a common cardiovascular disease. In China, VMC cases have been increasing year after year. VMC has become one of the main causes of sudden unknown death events in teenagers. Moreover chronic VMC often results in dilated cardiomyopathy. These demonstrate that VMC has greatly threatened human health. However, up to now, the mechanism of VMC has been unclear, which leads to the lack of effective treatments of VMC. So it has importantly theoretic and practical significance to elucidate the mechanism of VMC.It is known that viral infection is an important pathogeny of VMC, and VMC caused by coxsackievirus group B type 3 (CVB3) infection accounts for more than fifty percent VMC cases. Previous studies showed that CVB3 could not only injury cardiac myocytes by Pro2A directly, but also induce indirect injury by non-specific and specific immune responses. It is well known that indirect immunological injury in VMC is associated with the infiltration of a great amount of inflammatory cells. The infiltration of inflammatory cells is one of characteristic pathological changes in VMC. However, no reports were found about how the infiltration of inflammatory cells was triggered and how inflammatory cells arrested in tissue to induce immunological injury. It would open a new window in understanding the pathogenesis of VMC to resolve these questions.Generally, the infiltration of inflammatory cells is a complex and multiple-stepped process involved in many cells and molecules. Induced by inflammatory signals and cell-adhesion molecules, various inflammatory cells accumulate in the site of infection. The process consists of rolling, adherence, extravasation and so on. It has been confirmed that chemokines (ChKs) can provide migrating signal, activate cells and initiate the trafficking of the inflammatory cells. Our previous study showed that after HBV infection, the expression level of hepatocyte house-keeping chemokine LARC changed, which could influence the infiltration of cytotoxic T lymphocytes (CTL). According to above, we hypothesized that CVB3 infection may change the expression of ChKs of myocardium, which provide migrating signal, activate the cells and initiate the trafficking of the inflammatory cells and then lead to VMC. <WP=13>Therefore, in this study we first established VMC models in vivo and in vitro. The expression of 19 characteristic ChKs in four ChKs subfamilies was detected by RT-PCR in order to screen the ChKs that may play an important role in VMC. Based on the results, we further studied the effects of CVB3 infection dose, infection time and CVB3 state on the expression of screened ChKs. Then by increasing expression level of the ChKs in myocardium or intervening with the ChKs, we studied the role of the ChKs in pathogenesis of VMC. Our study would provide new theoretic base and experimental evidence for prevention and therapy of VMC.PARTⅠ Expression profile of ChKs in myocardial tissue of VMC1. Expression profile of ChKs in myocardial tissue of VMC in vivoThe chemokine superfamily is divided into four subfamilies (CXC, CC, C, CX3C) based on the arrangement of their amino terminal cysteine residues. To date, over 50 chemokines and 19 chemokine receptors have been identified. In order to screen the ChKs that may play an important role in VMC, the expression of 19 ChKs was detected by RT-PCR in myocardial tissue of BALB/c mice that were inoculated intraperitoneally with CVB3. The results showed that 13 ChKs including MIP-2, MIG, IP-10, SDF-1 (CXC family), MIP-1β, MCP-1, MCP-2, MCP-3, MCP-5, MDC, RANTES (CC family), LTN (C family) and FKN (CX3C family) were detectable in the myocardial tissue of VMC mice, whereas only 10 ChKs including SDF-1, MIP-1β, MCP-1, MCP-2, MCP-3, MCP-5, MDC, RANTES, FKN, LTN were found in normal control. Six ChKs (BLC, BRAK, MIP-1α, Eot, LARC and ALP) were detected neither in VMC group nor in normal control. It indicated tha更多还原